Carbamyl phosphate synthetase is an important enzyme in bacteria and eukaryotes, supplying an essential intermediate for both arginine and pyrimidine biosynthesis. The sequences of the small and large subunits of E. coli and yeast carbamyl phosphate synthetase have clearly shown that the large subunit catalyzing the synthesis of carbamyl phosphate from NH3 has evolved as a result of a duplication/fusion of an earlier gene. A comparative analysis of the primary sequences with other known amidotransferases and kinases have also uncovered evolutionary kinships hitherto unsuspected. Several possible binding sites for ATP have been identified in the large subunit. Furthermore, a short sequence common to many amidotransferases is also found in the small subunit and is presumed to be involved in glutamine hydrolysis. The present application addresses itself to a further exploration of the evolution and catalytic properties of this important enzyme. Specifically, studies will be undertaken to ascertain whether the large subunit evolved from the more primitive carbamate kinase. The genes for several enzymes that utilize acetylglutamate either as an allosteric activator (carbamyl phosphate synthetase) or as a substrate (acetylglutamate kinase) will be cloned and sequenced in order to identify the binding domain for this substrate. The sequences may provide a basis for further elucidating the mechanism by which new substrate and cofactor sites are acquired in complex enzymes. Finally, specific sequences considered to enhold regulatory and catalytic sites of the enzyme will be cloned and expressed in E. coli. Antibodies to the cloned protein domains will be employed to further confirm the functional domains of carbamyl phosphate synthetase.